The nucleus, like all cellular organelles, is composed of a unique set of proteins responsible for its structure and function. The long-term objective of the proposed research is to develop a detailed understanding of the mechanisms of assembly of proteins into the nuclear structure from their site of synthesis in the cytoplasm. This problem is being studied both biochemically and genetically in the yeast, Saccharomyces cerevisiae. Proteins important for nuclear assembly have been identified by isolation of a new class of yeast mutants, termed npl for nuclear protein localization. An in vitro nuclear protein assembly reaction has been developed and two proteins important for localization have been identified (termed NLS binding proteins). In this proposal, experiments designed to further investigate the role of these proteins, and identify other components such as nuclear pore proteins, important for nuclear protein localization are described. The specific aims are: 1) to characterize the NPL1 gene product with respect to its intracellular location and to determine with what other proteins it may interact, 2) to further characterize genetically and biochemically npl2 and npl3 mutants, 3) to purify and determine the precise role of the two NLS-binding proteins, 4) to use our in vitro nuclear assembly reaction to define other components necessary for import of proteins into the nucleus, and 5) to isolate and characterize additional yeast mutants defective in nuclear protein localization. The detailed picture of how proteins localize to the nucleus that should emerge from these experiments will aide our understanding of how eukaryotic viruses assemble in the nucleus. The ability to target proteins to certain sub-cellular compartments, such as the nucleus, may also have potential therapeutic value.